Pyrroloquinoline quinone (PQQ) from methanol dehydrogenase and tryptophan tryptophylquinone (TTQ) from methylamine dehydrogenase

PYRROLOQUINOLINE QUINONE (PQQ) FROM METHANOL DEHYDROGENASE AND TRYPTOPHAN TRYPTOPHYLQUINONE (TTQ) FROM METHYLAMINE DEHYDROGENASE BY VICTOR L. DAVIDSON Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi 39216

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Pyrroloquinoline Quinone (PQQ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II1. Methanol Dehydrogenase (MEDH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Structural Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Steady-State Kinetic Mechanism of MEDH . . . . . . . . . . . . . . . . . . . . . . . . . . C. Specific Effects of Ammonia on MEDH Activity . . . . . . . . . . . . . . . . . . . . . . D. Roles of Calcium in MEDH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Spectroscopic and Redox Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E Chemical Reaction Mechanism for Methanol Oxidation by MEDH . . . . . . G. Electron Transfer from MEDH to c-TypeCytochromes . . . . . . . . . . . . . . . . H. Biosynthesis of PQQ and MEDH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . W. Tryptophan Tryptophylquinone (TTQ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Methylamine Dehydrogenase (MADH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Structural Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Spectroscopic and Redox Properties of MADH . . . . . . . . . . . . . . . . . . . . . . C. Chemical Reaction Mechanism of MADH . . . . . . . . . . . . . . . . . . . . . . . . . . . D. TTQ in Electron Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Roles of pH and Cations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E Biosynthesis o f T T Q a n d MADH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Summary and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

95 96 98 99 100 102 104 1(15

1~6 109 111

I 12 I 12 113 1I.4 119 126 133 133 135

136

I. INTRODUCTION Pyrroloquinoline quinone (PQQ) and tryptophan tryptophylquinone ( T T Q ) have s i m i l a r s t r u c t u r a l f e a t u r e s (Fig. 1) a n d for a t i m e T T Q d e p e n d e n t e n z y m e s w e r e i n c o r r e c t l y b e l i e v e d to possess P Q Q . D e s p i t e t h e i r s t r u c t u r a l similarities, it is n o w e v i d e n t t h a t t h e s e c o f a c t o r s are d e r i v e d b y c o m p l e t e l y d i f f e r e n t m e c h a n i s m s . P Q Q is s y n t h e s i z e d e x o g e n o u s l y a n d t h e n associates with a n a p o p r o t e i n to f o r m t h e active h o l o e n z y m e . T T Q is f o r m e d via p o s t t r a n s l a t i o n a l m o d i f i c a t i o n of-two t r y p t o p h a n residues of the polypeptide chain. M t h o u g h each provides a reactive c a r b o n y l at t h e e n z y m e active site, P Q Q a n d T T Q h a v e differe n t catalytic f u n c t i o n s i n t h e i r r e s p e c t i v e e n z y m e s . P Q Q p a r t i c i p a t e s 95 .4DI~4N(~E.'~ LV PIfOTI'fI~, ' (JfI:'),tf~;TR}: l'(d. 58

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96

V I C T O R L. DAVIDSON

COOH

~R

H0 0 C//L"~- N~ ~ 0 0 0 PQQ

TTQ

FIG. 1. The structures of pyrroloquinoline quinone (PQQ) and tryptophan tryptophylquinone (TI'Q). In TTQ, the two sites of covalent attachment to the polypeptide chain are indicated as R.

primarily in the enzyme-catalyzed oxidation of alcohols and sugars, whereas T T Q participates in the enzyme-catalyzed oxidative deamination of amines. Both cofactors function in NAD(P)+-independent dehydrogenases that use other redox proteins as electron acceptors. In contrast to oxidases and pyridine nucleotide-dependent dehydrogenases, the reoxidation of the cofactor in PQQ and TTQ enzymes must occur via long-range surface-mediated electron transfer to an electron acceptor, rather than direct transfer at the enzyme active site. As such, P Q Q and TTQ enzymes have an interesting c o m m o n mechanistic feature of coupling active-site chemistry to surface-mediated electron transfer reactions. This review will focus on two well-characterized representative members of each class of these enzymes, the PQQ-dependent methanol dehydrogenase (MEDH) and the TTQ-dependent methylamine dehydrogenase (MADH).

II. PYRROLOQUINOLINEQUINONE(PQQ) Although the nature of its cofactor was not known at the time, the first PQQ-dependent enzyme to be characterized was a pyridine nucleotide-independent bacterial glucose dehydrogenase reported by

PQQANDTTQ

97

Hauge (1964). The enzyme possessed a dissociable cofactor that was neither a pyridine nucleotide nor a flavin. This cofactor had unpreced e n t e d properties and it was suggested that it could be a substituted n a p h t h o q u i n o n e . At approximately the same time, a key enzyme in bacterial methanol metabolism, methanol dehydrogenase (MEDH), was also shown to possess a dissociable organic cofactor that was neither a pyridine nucleotide nor a flavin. Based on its fluorescence properties, Anthony and Zatman (1964) proposed that this cofactor might be an unusual pteridine. For the next decade the status of the identity of these unusual cofactors remained unchanged. In 1979, the precise structure of this cofactor was deduced by Salisbury et al. (1979) fi-om Xray diffraction studies of a crystalline acetone adduct of the cofactor front MEDH. The structure was that of 4,5-dihydro-4,5-dioxo-l-Hpyrrolo [2,3-f]quinoline-2,7,9-tricarboxylic acid (Fig. 1). For a brief time this cofactor was referred to by the trivial name, methoxatin, h is now most commonly called pyrroloquinoline quinone or PQQ. Once the structure and certain properties of PQQ were known, it became possible to design analytical methods to detect PQQ in proteins and biological fluids. Many of these methods were based on comparisotl of dissociable cofactors or derivatives of such molecules with authentic PQQ and derivatives. Furthermore, it was demonstrated that PQQ could reconstitute the biological activity of an apoenzyme form of glucose dehydrogenase (Ameyama et al., 1985). Reconstitution was possible with either the purified apoenzyme or with crude membrane preparations that contained the apoenzyme. This finding was the basis fgr the development of a biological assay to identify and quantitate the presence of h'ee PQQ in biological fluids and in extracts of proteins or cells. Using this method t0r detection of PQQ, a wide range of bacterial e n ~ m e s were shown to possess noncovalently bound PQQ as a cofactor. All are wridine nucleotideindependent dehydrogenases. Glucose and methanol dehydrogenases front several diverse bacterial species were shown to possess PQQ (reviewed in Anthony, 1986, 1993; Matsushita and Adachi, 1993a; Goodwin and Anthony, 1998). In addition to these, many other pQQ-dependent en~a'nes have been characterized that use as substrates a range of alcohols, sugars, and aldehydes (reviewed in Matsushita and Adachi, 1993a, 1993b; Goodwin and Anthony, 1998). With the aldehyde dehvdrogenases, it is likely that the hydrate (the gem diol) is the true substrate. The best characterized of these PQQ-dependent enzymes are listed in Table I. The characterization of enzymes as PQQ dependent was not without controversy. Using a flawed hydrazine method of van der Meer el (d. (1987), several enzymes were incorrectly determined to possess PQQ. This was particularly problematic with enzymes that contained covalen~lv

98

VICTOR L. DAVIDSON TABLE l

Classes of PQQ-Dependent Enzymes a

Enzyme

Prosthetic

Cell

groups

location

Methanol dehydrogenase PQQ

Organisms

Periplasm Methylotrophicand autotrophic bacteria Periplasm Pseudomonasaeruginosa, P. putida

Alcohol dehydrogenase (type I)

PQQ

Alcohol dehydrogenase (type II) Alcohol dehydrogenase (type III) Glucose dehydrogenase Glucose dehydrogenase

PQQ, heine c PQQ, heme c PQQ PQQ

Periplasm

Comamonas testosteroni, P. putida

Membrane

Gluconobacteg, Acetobacter

Periplasm Membrane

Acinetobacter calcoaceticus

Aldehyde dehydrogenase

PQQ

Membrane

Acetic acid bacteria

A. calcoaceticus, E. coli, K. pneumoniae, Gluconobacteg, Pseudomonas sp.

a References for individual enzymes isolated from specific species of bacteria may be found in the following reviews: Anthony (1986), Masushita et al. (1994), and Davidson (1993).

b o u n d prosthetic groups. Many of these enzymes were subsequently shown to possess other novel covalently b o u n d cofactors, including TTQ, as well as amino acid-based free radicals that participate in catalysis (Davidson, 1993; Klinman, 1995). To date no enzyme has been shown to possess covalently b o u n d PQQ. Solid evidence has been obtained that P Q Q is an important and possibly essential factor for the proper growth and development of mammals (Killgore el al., 1989; Smidt et al., 1991a, 1991b; Kumazawa et al., 1992; Gallop et al., 1993). In addition to the biological assay for P Q Q determination, analytical methods that employ mass spectrometry (Suzuki and Kumazawa, 1997) and monoclonal antibodies specific to P Q Q (Narita and Morishita, 1997) have verified the presence of P Q Q in mammalian tissues and fluids. Despite these findings, to date no enzyme from a eukaryotic source has been shown to utilize PQQ. Thus, it is clear that P Q Q is a widely distributed and important cofactor in prokaryotic systems, but whether its distribution extends to enzymes of higher organisms remains to be proved.

III. METHANOLDEHYDROGENASE(MEDH) MEDH catalyzes the first step in the process of bacterial methanol metabolism. The realization in the late 1960s that several bacterial

PQQ AND TTQ

9~1

species were capable of growth on either methane or methanol as a sole source of carbon stimulated interest not only among microbial physiologists, but also among commercial interests that h o p e d to develop costeffective fermentation processes that were based on methylotrophic bacteria. MEDH has been isolated from several methylotrophic and autotrophic bacteria (Anthony, 1986, 1993). The amino acid sequences and X-ray crystal structures have been determined for some of these enzymes. The genetics of the biosynthesis of MEDH and P Q Q is con> plicated and has been worked out in three organisms. This section reviews the structural features of MEDH, the kinetic and chemical reaction mechanisms by which it oxidizes primary alcohols, the mechanism by which it transfers electrons to its physiologic electron acceptor, which is a c-type cytochrome, and novel features of its biosynthesis.

A. Structural Studies High-resolution crystal structures have been reported for the MEDHs from Methylobacterium extorquens AM1 (Ghosh et al., 1995) and from Methylophylus methylotrophus W3A1 (Xia et al., 1996). The structures are very similar and contain several interesting features. MEDH exhibits an 0t2~2 structure of larger 66 kDa PQQ-binding subunits and smaller 8.5 kDa subunits (Fig. 2). The function of the smaller subunit is not known. The 0t subunit of MEDH exhibits an eightfold radial symmetry c o m p o s e d of eight antiparallel four-stranded ~ sheets. This structure is stabilized by an unusual tryptophan docking motif (Ghosh et al., 1995) involving a repeating pattern of tryptophan residues that are located on the outer strands of the ~ sheets. P Q Q is located on the pseudo eightfold rotation axis of the 0t subunit, at the base of a tim_ nel-shaped cavity. The PQQ-binding site contains some particularly interesting features that are likely to be important for the catalytic and redox properties of P Q Q in MEDH. P Q Q is sandwiched between the indole ring of a tryptophan residue and a very unusual disulfide b o n d that is formed by two adjacent cysteine residues (Fig. 3). The indole ring of' the tryptophan is nearly coplanar with PQQ. This tryptophan and the disulfide: are each within 4 A of PQQ. The P Q Q cofactor is also coordinated with Ca 2+ in the active site (Fig. 4), The Ca 2+ is hexacoordinate with three of the ligands provided by the 0 5 , N6, and O7A of PQQ. The other three ligands are provided by active-site residues, the two side chain oxygens of a glutamate, and one side chain oxygen of an asparagine. The possible relevance of these structural properties to the catalytic and electron transfer properties of MEDH will be addressed.

] 00

VICTORL. DAVIDSON

a subunits

J 13 subunits FIG. 2. The structure of methanol dehydrogenase. The structure is that of the enzyme from M. methylotrophusW3A1 (Xia et al., 1996; Protein Data Bank entry 1B2N). Only the protein backbone is shown. The larger ¢x subunits are black and the smaller ~ subunits are gray.

B. Steady-State Kinetic Mechanism of M E D H M E D H catalyzes t h e o x i d a t i o n o f a p r i m a r y a l c o h o l to its c o r r e s p o n d i n g a l d e h y d e , a n d i n t h e p r o c e s s t r a n s f e r s two e l e c t r o n s f r o m t h e s u b s t r a t e to s o m e e l e c t r o n a c c e p t o r [A i n Eq. (1) ].

C104

W237 Fro. 3. Interactions of PQQ with a tryptophan and the vicinal disulfide bond in methanol dehydrogenase. The structure and residue numbers are those of the enzyme from M. methylotrophusW3A1 (Xia et al., 1996; Protein Data Bank entry 1B2N).

PQQ AND TTQ

101

QQ

R324

D297

Flc;. 4. Interactions of Ca > with P Q Q and amino acid residues in the active site of methanol dehydrogenase. The structure and residue numbers are those of the enzyme f r o m M. methylotrophus W3A1 (Xia et al., 1996; Protein Data Bank entry 1B2N). The dashed lines indicate the interaction of Ca > with its ligands.

RCH2OH + 2Aox -+ RCHO + 2Ar~.a+ 2H +

(1)

This reaction is the first step in the metabolism of methanol, which can serve as a sole source of carbon and energy for bacteria that possess this enzyme (Anthony, 1986). When MEDH is assayed in vitro, small redoxactive species such as phenazine methosulfate or Wurster's blue are routinely used as the electron acceptor (Frank and Duine, 1990). The natural electron acceptor for MEDH is a c-type cytochrome, cytochrome q~ in many methylotrophic bacteria (Anthony, 1992) and cytochrome c-551i in Paracoccus denitrificans (Husain and Davidson, 1986). The mechanism of the reductive half-reaction of MEDH and the conditions used in the steady-state assay of its activity have been the topic of much controversy over the past 30 years. The in vitro activiw of MEDH with artificial electron acceptors requires ammonia and cyanide, but it is not clear why. Furthermore, although ammonia and cyanide are activators of MEDH at low concentrations, each acts as an inhibitor at higher concentrations (Harris and Davidson, 1993a). A kinetic model that was proposed to describe the peculiar steady-state kinetic behavior of P. denitrificans MEDH is shown in Fig. 5 (Harris and Davidson, 1993a). In model studies, it has been demonstrated that the C5 carbonyl of PQQ is reactive

102

VICTOR L. DAVIDSON

e

<

Esemi

~

Ere d

S Eox-NH 2

E0 X

~

E o~ -S

'~

NH 3

CN S

E ox -NH 2

<~

E

NH 3

ii

OX

CN

I E ox-S

I/

C N-

>

Ered-P

N!

E OX-CN FIG. 5. Proposed steady-state kinetic mechanism for the reaction cycle of methanol dehydrogenase. This mechanism was proposed by Harris and Davidson (1993a) for the P denitrificansenzyme. E* represents the cyanide-activated form of MEDH. S and P represent, respectively, methanol substrate and formaldehyde product.

with nucleophiles a n d forms adducts with m e t h a n o l , aldehydes, cyanide, and a m m o n i a (Itoh et al., 1993). This supports the n o t i o n that adducts with these c o m p o u n d s may play roles in the chemical reaction mechanism o f MEDH. However, n o b o d y has yet r e p o r t e d the isolation of a covalent a m m o n i a or cyanide a d d u c t o f M E D H so there is n o direct evidence that e i t h e r effector binds directly to PQQ, o r to a m i n o acid residues in the active site, or at an allosteric site.

C. Specific Effects of Ammonia on M E D H Activity 1. Activation by Ammonia Kinetic studies with M E D H f r o m Hyphomicrobium X indicated that the m a g n i t u d e o f the d e u t e r i u m kinetic isotope effect for the steady-state r e a c t i o n d e c r e a s e d f r o m 6.7 to 1.4 as the c o n c e n t r a t i o n o f NH4C1

eQQANDTTQ

103

increased from 1.25 mM to 40 mM (Frank et al., 1988). This result suggested that a m m o n i a facilitated the transfer of hydrogen from methanol to PQQ. Therefore, the model in Fig. 5 includes a role for a m m o n i a in activating the conversion of the cyanide-activated, oxidized enzyme-substrate complex, to the reduced enzyme-product complex. 2. Inhibition by Ammonia The inhibition of MEDH by ammonia is due to a process that is completely distinct from the process of activation by ammonia. The inhibition most likely involves covalent adduct formation with PQO~ given the observed spectral perturbations caused by ammonia in the concentration range of the Ki for ammonia. It is known that ammonia reacts with h'ee PQQ in solution to form an iminoquinone (Duine et al., 1987). Forrest et al. (1980) proposed that this may be an intermediate in the reaction mechanism with the alcohol substrate. Such a mechanism of activation, however, has been discounted primarily because no perturbations of the absorption spectrum of MEDH were observed on addition of ammonia at concentrations sufficient to cause activation of the enzyme. The observation that perturbations of the absorption spectrum of the enzyme do occur on addition of ammonia, but only at the much higher concentrations that correlate with inhibition of activity (Harris and Davidson, 1993a), provides strong evidence that an iminoquinone intermediate is not necessary for catalysis, but in fact may prevent catalysis. It follows that the previously discussed activation bv ammonia must be a result of its interaction with MEDH at a different site than PQQ. While the actual mechanism of inhibition by ammonia is not known, additional evidence for iminoquinone formation has been obtained from model studies of the reaction of PQQ trimethylester with ammonia in organic solvents (Itoh et al., 1991). The visible spectra of the quinone and the iminoquinone are similar but the latter exhibits a slightly e n h a n c e d absorbance in the range from approximately 350 to 430 nm with an isosbestic point at approximately 430 nm. These spectral changes on iminoquinone formation are similar to what has been observed on addition of ammonia to MEDH. Other explanations for these spectral perturbations, however, cannot be ruled ont. For example, a m m o n i u m ion may also be acting as a counterion to oxyanions in the active site and in doing so perturbs the microenvironment of the cofactor in such a way as to alter the spectrum. Thus, until a covalent adduct of MEDH with ammonia can be isolated and characterized, the precise molecular basis for the effects of ammonia on MEDH activity ren~ains uncertain.

104

VICTOR L. DAVIDSON

D. Roles of Calcium in MEDH The presence of Ca 2+ at the active site is not unique to MEDH among quinoproteins. It appears that a c o m m o n feature of all PQQ-dependent enzymes is a requirement for calcium or some divalent cation. In some enzymes the cation is more tightly b o u n d than in others, but in MEDH it cannot be removed without irreversibly denaturing the enzyme (Richardson and Anthony, 1992). To examine the role of Ca 2+ in MEDH, calcium was replaced with strontium in the P denitrificans enzyme and with strontium or barium in the M. extorquens enzyme. With P. denitrificans MEDH, it was possible to replace Ca 2+ with Sr 2+ by growing the cells in media in which calcium salts had been replaced with strontium salts (Harris and Davidson, 1993b). A n t h o n y and co-workers were able to produce active M. extorquens MEDH in which Ca 2+ was replaced with either Sr 2+ or Ba 2+ by a different procedure (Richardson and Anthony, 1992; Goodwin and Anthony, 1996). They isolated metal-free MEDH from an mxaAm u t a n t strain of the bacterium. The mxaA gene is required for Ca 2+ insertion into MEDH during biosynthesis. Incubation of the apoenzyme with the alternative divalent cation led to formation of active metal-substituted enzyme. The Sr2+-containing P. denitrificans MEDH exhibited an increased extinction coefficient for the P Q Q chromophore, and displayed certain kinetic properties that were different from native MEDH (Harris and Davidson, 1993b). Replacement of Ca 2+ with Sr 2+ also increased the thermal stability of P denitrificans MEDH (Harris and Davidson, 1994). The BaZ+-containing M. extorquens MEDH exhibited a lower activation energy for oxidation of methanol and higher Vr~ax than CaZ+-MEDH, but also exhibited a much higher Km for methanol and KA for ammonia as an activator. These changes were attributed to a change in conformation of the active site caused by the larger Ba 2+ atom (Goodwin and Anthony, 1996). Together these data suggest that Ca 2+ plays important roles both in catalysis and in stabilizing the structure of MEDH. Itoh and co-workers examined the effect of Ca 2+ on the reactions catalyzed by P Q Q model compounds, such as P Q Q trimethylester (Itoh et al., 1997, 1998). They d e m o n s t r a t e d that Ca 2+ f o r m e d a complex with the P Q Q model c o m p o u n d s that facilitated alcohol adduct formation at the C5 position of PQQ. Complex formation with Ca z+ was also required for the base-catalyzed oxidative elimination of the adduct that could be observed in the presence of a strong base. These results also support the contention that the active-site Ca 2+ in MEDH not only plays an important structural role but also plays a direct role in catalysis.

PQQANDTTQ

1.6

'A

1.2

O

O t-

1

0.15

/

'\j'/

\

B

/

i,I /

0.10

~ 0.8 0 m

<

0.20

105

',,,

0.05

0.4 0.0 250

,

,

300

350

~

400

Wavelength (nm)

\,

0.00

450

,

300

350

.

400

450

Wavelength (nm)

Flc. 6. Absorption spectrum of methanol dehydrogenase. (A) The UV/visible spectrum of the enzyme as isolated from P. denitrificans in 0.1 M potassium CHES (2-cych)hexylamino-ethanesulfonic acid), pH 9.0. (B) Magnification of the portion of the spectrum that is attributed to the PQQ chromophore.

E. Spectroscopic and Redox Properties 1. General Features T h e absorption spectrum o f P. denitrificans MEDH, as isolated, is shown in Fig. 6. All MEDHs exhibit a similar spectrum that is believed to be that o f the s e m i q u i n o n e r e d o x form. T h e spectrum does n o t change on addition o f substrate because the enzyme must be first oxidized to react with methanol. T h e r e d o x potential of free P Q Q has b e e n determined. It is +90 mV (versus SHE) at p H 7.0 for the P Q Q / P Q Q H 9 couple and varies with p H as e x p e c t e d for a two electron-two p r o t o n r e d o x carrier (Duine et al., 1981). T h e r e d o x potential for M E D H has not b e e n reported, and it has not b e e n possible to generate a stable oxidized or r e d u c e d form of M E D H in vitro. This is at least in part due to the curious feature that all MEDHs are able to u n d e r g o a steady-state reaction with an electron acceptor in the absence o f a d d e d substrate (Anthony and Zatman, 1964; Duine and Frank, 1980; Ghosh and Quayle, 1981; Harris and Davidson, 1993a). This has b e e n attributed to the presence o f an " e n d o g e n o u s substrate," which has never b e e n identified. T h e M E D H s e m i q u i n o n e may be oxidized by Wurster's blue, the perchlorate salt of the cationic free radical of N,N,N;N;-tetramethyl-p-phenylenediamine. However, the oxidized enz~ane is t h e n apparently immediately r e d u c e d by the e n d o g e n o u s substrate, and t h e n s o m e h o w quickly reverts to the s e m i q u i n o n e form. T h e r e is some evidence that the s e m i q u i n o n e form is g e n e r a t e d via reaction with oxygen (discussed in Section III, E, 2). In any case, n o o t h e r spectral forms

106

VICTOR L. DAVIDSON

of the enzyme are observed. This inability to characterize the spectra of the different redox forms of MEDH has made it very difficult to obtain mechanistic information.

2. Possible Role of Oxygen is Stabilizing the MEDH Semiquinone The reduced and semiquinone forms of free P Q Q a r e known to react with 02 to form superoxide anion (02-) in solution (Duine et al., 1987). However, all of the enzymes that are known to utilize P Q Q are dehydrogenases. The activity ofP. denitrificans MEDH with different electron acceptors was compared u n d e r aerobic and anaerobic conditions, and in the presence and absence of agents such as superoxide dismutase. Evidence was obtained that u n d e r certain conditions O2 reacted with reduced forms of the protein-bound P Q Q to generate superoxide (Davidson et al., 1992). These results were the first to suggest that an enzyme-bound P Q Q cofactor could react directly with 02. The proposed mechanism for this interaction is shown in Eq. (2). It suggests that the latter two reactions [(2b) and (2c)] are normally at equilibrium, and do not result in a net turnover of the redox state of the enzyme. These reactions with oxygen may explain the peculiar redox properties of MEDH, its apparent isolation as a stable semiquinone species, and the lack of spectral change on addition of methanol. E-PQQ + CH3OH ---) E-PQQH2 + CH20 E-PQQH2 + 02 +-) E-PQQH. + H + + 02E-PQQH. + 02 ~ E-PQQ + H + + 02-

(2a) (2b) (2c)

3. Stabilization of the Oxidized Redox Form of Ba2+-Substituted MEDH The spectrum of the oxidized redox form of the Ba2+-substituted M. extorquens MEDH has been reported by Goodwin and Anthony (1996). Because of the low affinity for substrates of Ba2+-substituted MEDH relative to that of the native Ca2+-MEDH, it was possible to observe the oxidized enzyme and monitor its relatively slow reduction after rapid removal of Wurster's blue. Relative to the spectrum shown in Fig. 6, the oxidized Ba2+-MEDH exhibited a bleaching of the peak centered around 350 nm and increase in the broad peak centered around 400 nm. This important study has provided the most convincing evidence thus far as to the true nature of the features of the UV/visible absorption spectrum of the oxidized form of MEDH.

F. Chemical Reaction Mechanism for Methanol Oxidation by MEDH For this extensively studied P Q Q - d e p e n d e n t enzyme, kinetic analyses, use of the mechanism based-inhibitor cyclopropanol, and studies

PQQAND TTQ

107

A

4

Ca2÷

O ~ , H3

Ca2+

%0. .~0

Ca2+

2NO 3Hc / O ~ H

.~_

"---

Asp

~---

~.

Asp

Asp

B

COOH

HOOC

~

H~N/ __ ~-

HOOC'/&~N-o/~C~

H ~ HO

HOOC ~

"N"

COOH H+ /

~ "O-O'A0 . %_ "'~ ~CH 3

F[(;. 7. (A) Proposed hemiketal chemical reaction mechanism for the oxidation of methanol by methanol dehydrogenase. Only the quinone portion of PQQ is shown with the C4 and C5 carbons indicated. (B) Proposed role for the pyrrole nitrogen of PQQ in the ionization of the C4 oxygen in the hemiketal intermediate.

with PQQ model compounds have provided considerable insight into the reaction mechanism of MEDH. However, critical questions remain unresolved and it has not yet been possible to definitively answer the question of whether a covalent intermediate is part of the chemical reaction mechanism. There is still debate as to whether the oxidation of methanol involves the formation of a covalent hemiketal adduct between P Q Q and methanol, or whether it proceeds via a simple hydride transfer mechanism. In the hemiketal mechanism (Fig. 7A) an active-site base, believed to be an aspartic acid residue, abstracts a proton from methanol to yield a methoxy anion that initiates a nucleophilic attack of the C5 of PQQ. A methyl proton is then abstracted by the C4 oxygen in the hemiketal intermediate concomitant with release of the aldehyde product and formation of the P Q Q quinol. This mechanism was first proposed by Frank et al. (1989). This mechanism was modified by Anthony (1996) to

108

VICTORL. DAVIDSON

Ca2,

Ca2*

2

0

>o

NO

>o Asp

Asp

x

Ca2*

>o .

Asp

FIG. 8. Proposed hydride transfer chemical reaction mechanism for the oxidation of methanol by methanol dehydrogenase. Only the quinone portion of PQQ is shown.

include a role for the pyrrole nitrogen atom of PQQ. This allows ionization of the C4 oxygen in the hemiketal intermediate, which facilitates the proton abstraction from the methyl carbon (Fig. 7B). In the hydride transfer mechanism (Duine et al., 1987) (Fig. 8) the active-site base again initiates the reaction by abstracting the alcoholic proton from the substrate. However, in this mechanism there is no covalent adduct formed between the substrate and PQQ. Instead, the substratederived hydride ion attacks the C5 carbon, resulting in formation of the aldehyde and reduction of PQQ. In either mechanism, the Ca ~+ at the active site may facilitate the reaction by enhancing the electrophilicity of the C5 carbon of P Q Q by way of its coordination with the C5 carbonyl oxygen. This would facilitate nucleophilic attack by either the substrate oxyanion or hydride ion. The results of studies of P Q Q model compounds in organic solvents suggest that the PQQ-catalyzed oxidation of methanol occurs via the covalent hemiketal intermediate. Itoh et al. (1997, 1998) demonstrated that a complex of the P Q Q model c o m p o u n d with Ca 2+ facilitated alcohol adduct formation at the C5 position of P Q Q and catalyzed the oxidative elimination of the adduct in the presence of a strong base. This mechanism is also consistent with the finding that tile reaction of MEDH with cyclopropanol yields a covalent propanal adduct at the C5 of P Q Q (Frank et al., 1989). The proposed reaction mechanism for the inactivation of MEDH by cyclopropanol is shown in Fig. 9. Conversely, theoretical ab initio computations that considered these alternative mechanisms for methanol oxidation by P Q Q / C a 2+ in the MEDH activesite environment suggested that the hydride transfer mechanism was more likely, due to the much larger energy barrier that was calculated

PQQ AND TTQ

"" B"

109

HO

BH

-~

"

C

H

O

B:

FIc;. 9. Proposed mechanism Ibr the inactivation of methanol dehydrogenase bx cyclopropanol. Only the quinone portion of PQQ is shown.

for the alternative addition-elimination method (Zheng and Bruice, 1997). A deuterium kinetic isotope effect of 6.7 in the presence of low concentrations of a m m o n i u m has been reported for the oxidation of methanol (Frank et al., 1988). This primary kinetic isotope effect, howeve~, is consistent with either of the two proposed mechanisms. As with MEDH, the nature of the reaction mechanism of another widely studied PQQ-dependent enzyme, glucose dehydrogenase, has been controversial. The crystal structure of glucose dehydrogenase has recently been determined with glucose b o u n d at the active site. Based on the position of the substrate relative to P Q Q and active-site amino acid residues, it was concluded that the oxidation of glucose most likely occurred via the general base-catalyzed hydride transfer mechanism rather than the covalent addition-elimination mechanism (Oubrie et al., 1999). Whether it follows that all PQQ-dependent enzymes follow the same mechanism is impossible to say at this time. However, these findings have served to rekindle the controversy concerning the mechanism of MEDH at a time when the consensus was beginning m favor the covalent hemiketal mechanism over the hydride transfer mechanism.

G. Electron Transfer from MEDH to c-Type Q~tochromes 1. Identit~ oJ the Natural Electron Acceptors The natural electron acceptors for MEDH are c-type cytochromes. In methylotrophic bacteria, such as M. extorquens, the acceptor is an acidic cytochrome qj, with L designating low isoelectric point (~Mathony, 1992). In P. denit~ficans, the acceptor has been designated cytochrome c-551i (Husain and Davidson, 1986). In each of these MEDH-cytochrome systems, it has been proposed that the cytochrome docks with MEDH in such a manner that the heine of the cytochrome is situated in close proximit?' to the funnel-shaped cavity that contains P Q Q (Harris and Dmidson, 1993c; Anthony et al., 1994; Dales and Anthony, 1995).

110

VICTORL. DAVIDSON

2. Kinetic and Thermodynamic Analysis of Electron Transfer MEDH and cytochrome c-551i from P. denitrificans must form, at least transiently, a complex to allow the physiologically relevant transfer of electrons from P Q Q to heine. The reoxidation of MEDH by the cytochrome was studied by stopped-flow spectroscopy (Harris and Davidson, 1993c) and the kinetic parameters for complex formation and electron transfer were examined as a function of ionic strength and temperature (Harris et al., 1994). Both the K~ for the MEDH-cytochrome complex and the rate constant for reduction of the cytochrome by MEDH (kET) varied with ionic strength. The observation that kET was dependent on ionic strength was unexpected. The variations of Ka and krT with ionic strength were each analyzed by Van Leeuwen theory (Van Leeuwen, 1983) to predict the orientations in which these macromolecules interact for binding and electron transfer, respectively. These analyses indicated that the optimal orientations for binding and electron transfer were similar but slightly different. These results were used to derive a model for "conformationaUy coupled" electron transfer (Harris et al., 1994), which describes the case where a relatively rapid but unfavorable rearrangement of the proteins after binding is required to produce the most efficient orientation for a relatively slow electron transfer reaction. Thermodynamic analysis of/Ca values obtained at different temperatures (Harris and Davidson, 1993c) indicated the importance of the hydrophobic effect in complex formation. Analysis of the temperature d e p e n d e n c e of kET by electron transfer theory (the theory is discussed in detail in Section V, D, 1) predicted an electron transfer distance of approximately 15 fk. Analysis of the crystal structure of MEDH revealed that the minimum distance from P Q Q to a surface accessible site is about 15 A, and in the crystal structure of cytochrome c-551i the heine is exposed at two sites on the protein surface. Thus, the distance predicted from the solution studies may be considered reasonable based on this structural information and consistent with the proposed docking site for the cytochrome with MEDH.

3. Possible Roles of the Unusual Vicinal Disulfide Bond in MEDH The position of the unusual vicinal disulfide b o n d in MEDH (see Fig. 3) suggests the possibility that it may be involved in electron transfer from P Q Q to the cytochrome c electron acceptor, which is believed to dock with MEDH at the protein surface in the vicinity of that bond near the top of the funnel-shaped cavity that contains PQQ. Avezoux et al. (1995) demonstrated that reduction of this disulfide bond rendered MEDH inactive in electron transfer to the cytochrome. However, carboxymethylation of the thiols after reduction led to the restoration of

PQQANDTTQ

lll

TABLEII ProposedPolypeptidePrecursorsof PQQ Organism

Sequence"

Acinetobacter ca&oaceticus

M___Q..~_'TK12AFTDLRIGFEVTMYFEAR

Goosen et al., 1989

Kb,bsiella pneumoniae

M-~Aq~KPAFIDLRL~[~VTI._YISNR

Meulenberg et al.. 1992

Methylobacterium extcqquens

MKWAAP~ZSEICVGME\q'SYESAI~21DTFN Morris el al., 1994

Pseudomonas fluorescens Methylobacillus flagellatum

MYRQHPSHPPQRSNFM'I~SKI2AYTDLRIGFE\q'MYRASR MMD,"TKPEVI'EMRF(;FE\,~FMYVCNR

Reference

Schnider el al., 1995 (;omelskv el al., 1996

" T h e residues that are conserved in each polypeptide are underlined and tile glutamic acid and tyrosine residues that are believed to be the precursors for tile biosynthesis of PQQ are indicated in bold type.

this activity. It was concluded, therefore, that the disulfide does not function as a redox-active intermediate in the electron transfer to the cytochrome. It is noteworthy that this disulfide bond is not conserved in the structurally similar P Q Q - d e p e n d e n t glucose dehydrogenase (Oubrie et al., 1999). It has been suggested that this unusual structural feature may be involved in the stabilization of the semiquinone form of PQQ (Avezoux et al. 1995). Alternatively, the disulfide bond may serve to prevent dissociation of P Q Q from the enzyme. This is consistent with the observation that P Q Q is more easily removed fiom glucose dehydrogenase and may be reconstituted with the apoenzyme, whereas denaturing conditions are required to remove PQQ from MEDH.

H. Biosynthesis of PQQ and MEDH Nuclear magnetic resonance (NMR) studies of P Q Q biosynthesis in intact bacteria that were grown on ~3C-labeled amino acids revealed that each of the carbon atoms of PQQ was derived from tyrosine or glutamate, with each amino acid apparently incorporated intact (Houck et al., 1988, 1991). I n d e p e n d e n t of this, genetic studies revealed an essential gene for P Q Q biosynthesis that encodes a small peptide of 23 to 39 amino acids, depending on the bacterimn (Table II). These peptides possess a conserved tyrosine and glutamate. In Acinetobacter calcoaceticus, conversion by site-directed mutagenesis of either the conserved Glu-16 to aspartate, or Tyr-20 to phenylalanine, abolished P Q Q biosynthesis

112

VICTORL. DAVIDSON

(Goosen et al., 1992). While not absolute proof, these results have been taken as strong evidence that P Q Q is derived by posttranslational processing of this peptide precursor. As many as six genes are believed to be required for P Q Q biosynthesis. In addition to the unusual and complicated process of P Q Q biosynthesis, the assembly of MEDH is a remarkably complicated process. Genetic studies of different bacteria have identified a total of 32 genes that are required for methanol oxidation activity (Lidstrom et al., 1994). Three are structural: the two that encode the cx and [3 subunits of MEDH, and one that codes for the cytochrome electron acceptor of MEDH. Six are required for P Q Q biosynthesis and export into the periplasm. Three genes are required for proper insertion of calcium into MEDH. The other genes are either involved in regulation or have as yet unidentified roles. For a detailed review of the status of this area see Goodwin and Anthony (1998).

IV. TRYPTOPHANTRYPTOPHYLQUINONE(TTQ) T T Q is the prosthetic group of methylamine dehydrogenase (MADH). This enzyme was first characterized by Eady and Large (1968), but the exact nature of its prosthetic group remained unknown for several years. The difficulty in identification rested in large part from the fact that it was not dissociable from the protein even after denaturation. After the characterization of P Q Q as the cofactor of MEDH, there were suggestions that MADH also possessed a covalently b o u n d form of P Q Q or a P Q Q derivative. The structure of the prosthetic group of MADH was finally determined by McIntire et al. (1991) using chemical and NMR spectroscopic methods, to be 2",4-bitryptophan-6,7 dione. It was given the common name of tryptophan tryptophylquinone or TYQ. The structure was subsequently confirmed by X-ray crystallographic analyses of MADH (Chen et al., 1991). In addition to MADH, TI~Q has also been identified as the prosthetic group of aromatic amine dehydrogenase (Govindaraj et al., 1994). Remarkably, this prosthetic group is not acquired exogenously. It is formed via posttranslational modification of two tryptophan residues of the polypeptide chain (see Section V, F)

V. METHYl_AMINEDEHYDROGENASE(MADH) MADH is a periplasmic enzyme that has been purified from several gram-negative methylotrophic and autotrophic bacteria (Eady and Large, 1968; Shirai et al., 1978; Matsumoto et al., 1978; Kenny and McIn-

eQQAYDTTQ

113

tire, 1983; Vellieux et al., 1986; Husain and Davidson, 1987). It catalyzes the oxidative deamination of methylamine to formaldehyde plus ammonia, and in the process transfers two electrons from the substrate to some electron acceptor [A in Eq. (3) ]. CH:~NH:~+ + 2A~,x+ HzO --+ HCHO + NH, + + 2A,-,,t + 2H +

(3)

This reaction is the first step in the metabolism of methylamine, which can serve as a sole source of carbon and energy for these bacteria. When MADH is assayed in vitro, small redox-active species such as phenazine ethosulfate are routinely used as the electron acceptor (Davidson, 1990). The natural electron acceptor for most MADHs is a periplasmic type I "blue" copper protein, amicyanin, which mediates electron transfer from MADH to c-type cytochromes (Tobari and Harada, 1981; Lawton and Anthony, 1985; Husain and Davidson, 1985: van Houwelingen et al., 1985).

A. Structural Studies

The physical properties of the MADHs that have been characterized thus far indicate that they are a relatively well-conserved class of enzymes. Each MADH is a tetramer of two identical larger 0t subunits of molecular weight of 40,000 to 50,000, and two identical smaller 13 subunits of molecular weight of approximately 15,000 (Fig. 10). The smaller subunits each possess the covalently bound T T Q prosthetic group. Crystal structures have been determined for MADH from Parercoccus denitrificans (Chen et al., 1998), Thiobacillus versutus (Vellieux el al., 1989), and Methylobacterium extorquens AMI (Labesse et al., 1998). The TTQ-bearing subunits display a high level of structural similarity, as well as sequence homology (Chen et al., 1998). It is interesting to note that MADH shares a relatively unusual structural motif with MEDH. Like the larger 0t subunit of MEDH (see Section III, A), the larger 0t subunit of MADH exhibits a [~-sheet propeller-like pattern. In MADH, this is formed by seven four-stranded antiparallel 13 sheets (Chen et al., 1998). In MEDH, PQQ is located within the 0~ subunit on the eightfold pseudosymmetry axis. In MADH, T T Q is located on the 13subunit; however, its position is near the sevenfold pseudosymmetry axis projected into the ~ subunit. The active site of MADH is relatively hydrophilic and located at the end o f a hydrophobic channel between the o~and 13subunits. The C6 carbonvl of TTQ is exposed to solvent at the active site (Fig. 11), and is the site of covalent adduct formation with the substrate (Huizinga et al., 1992; Labasse el aL, 1998). The two indole rings that comprise the TTQ stnm-

114

VICTORL. DAVIDSON

0~ subunit

1~ subunit a subunit F~G. 10. The structure of methylamine dehydrogenase. The structure is that of the enzyme from P. denitrificans (Chen et al., 1998; Protein Data Bank entry 2BBK). Only the protein backbone is shown. The TTQ prosthetic groups on each ]3subunit are presented as space fill. The larger {xsubunits are black and the smaller ]3subunits are gray.

ture are not coplanar but at a dihedral angle o f approximately 38 ° (Chen et al., 1998). Whereas the C6 carbonyl o f T r Q is present in the active site, the edge o f the second indole ring, which does n o t contain the quinone, is exposed at the surface o f MADH. In addition to the structures o f free MADH, structures have also b e e n d e t e r m i n e d for P. denitrificans MADH in a binary protein c o m p l e x with amicyanin (Chen et al., 1992), and a ternary protein c o m p l e x with amicyanin and c y t o c h r o m e c-551i (Chen et al., 1994). Single crystal m i c r o s p e c t r o p h o t o m e t r y has b e e n used to d e m o n s t r a t e that the proteins in these crystallized complexes are capable o f catalysis a n d electron transfer (Merli et al., 1996).

B. Spectroscopic and Redox Properties of M A D H 1. C,eneral Features T h e study o f T T Q enzymes has b e e n facilitated by the fact that the r e d u c e d a n d s e m i q u i n o n e states o f these enzymes are relatively stable

PQQ AND TTQ

ASP 76

115

THR 122

Fl(;, l 1. The active site of methylamine dehydrogenase. The T F Q cotactor is black and residues that s u r r o u n d the cofactor are indicated. T h e dashed lines indicate hydr~gen b o n d i n g interactions between indole nitrogens on T T Q with main chain oxygen atoms otAla-103 a n d Set-30, and between the 0 7 of T T Q and the amide nitrogen of Asp32. The 0 6 is present at the e n d of a solvent accessible c h a n n e l and within hydrogen b o n d i n g distance of side chain oxygens of eksp-76 a n d Thr-122. The structure is that ot the enzyme fi-om P. denitrificans (Chen et al., 1998; Protein Data Bank e n u y 2BBK).

to reoxidation and that the quinone, semiquinone, and quinol redox states exhibit readily distinguishable visible absorption spectra (Davidson el al., 1995b) (Fig. 12). These absorption spectra are also quite distinct from other quinoproteins that possess either P Q Q or topaquinone as a prosthetic group. The fully reduced forms of MADH also exhibit a fluorescence spectrum that is absent in oxidized MADH (Eady and I,arge, 1971; Matsumoto, 1978). Excitation of reduced MADH at approximately 320 nm yields an emission maximum at approximately 380 mn due to fluorescence of the reduced T T Q cofactor. Two different forms of reduced and semiquinone MADH may be generated in vitro, which possess either oxygen or a substrate-derived nitrogen covalently bound to the C6 of T T Q (Fig. 13). The nonphysiologic Oquinol and O-semiquinone forms of MADH may be generated by reductire titration with sodium dithionite (Husain et al., 1987). The physiologic N-quinol form of MADH may be formed by reduction with substrate (Bishop et al., 1996b). The physiologic N-semiquinone of MADH may be formed by controlled light-induced oxidation of substrate-reduced MADH (Zhu and Davidson, 1998a). The ~4sible absorption spectra of the

116

VICTOR L. DAVIDSON

0.4

Qulnols Semiqulnones 0.3

e •

0

0O ~

0.2

0.1

0.0 300

I

I

400

500

600

Wavelength(nm) FIG. 12. Visible absorption spectra of different redox forms ofmethylamine dehydrogenase. All spectra were recorded in with 6.7 gM MADH in 10 mM BisTris propane (1,3bis[tris(bydroxyrnethylamino)]propane)buffer, pH 7.5, at 25°C. Spectra of the the O-quinone, O-semiquinone, and O-quinol are displayed as solid lines. Spectra of the Nsemiquinone and N-quinol are displayed as dotted lines.

O-forms and ~ f o r m s of MADH are very similar (Fig. 13) (Zhu and Davidson, 1999). In other words, whether O or N is b o u n d to C6 in the reduced and semiquinone redox states has minimal effects on the visible absorption spectrum of each of these redox forms of MADH.

2. Redox Properties of MADH The oxidation-reduction midpoint potential (F~) value for the twoelectron oxidized/reduced couple of P. denitrificans MADH has been determined by spectrochemical titration (Zhu and Davidson, 1998b). At p H 7.5 it is +95 mV (versus SHE, standard hydrogen electrode) and over the range of p H from 6.5 to 8.5 it is p H d e p e n d e n t and exhibits a change of approximately -30 mV per p H unit. This indicates that the two-electron transfer is linked to the transfer of a single proton. This result differs from what was obtained from redox studies of a T T Q

PQQ ANDTrQ

F•

jN

1 17

i•

__R

HO.,~~ ~ N

---

__R

e-

e-+H +

O-

O

O

O-quinol

jN

O-quinone

O-semiquinone

B -~

H

jN

i•

__R

O

N-quinol

e-+H* HN

jN

_R

eO

N-semiquinone

N-quinone

Fic;. 13. Sequential one-electron oxidations of dithionite-reduced (A) and substratcreduced (B) TTQin methylamine dehydrogenase. The protonation state of the oxygens in the quinol and semiquinone forms of TTQwas determined by redox studies (Zhu and Davidson, 1998b). The distribution of spin density in the semiquinone torms actually extends throughout the quinolated indole ring and into the second indole ring, but is asymmelric (Warncke el al., 1995; Singh et al., 2000) and should not be inl~q-red from this figure.

m o d e l c o m p o u n d , for which the two-electron c o u p l e is linked to the transfer o f two p r o t o n s (Itoh et al., 199.5). This result also distinguishes the r e d o x p r o p e r t i e s of the e n z y m e - b o u n d T T Q f r o m those of the m e m b r a n e - b o u n d q u i n o n e c o m p o n e n t s o f respiratory a n d p h o t o s y n t h e t i c e l e c t r o n transfer chains that transfer two p r o t o n s p e r two electrons. This d i l f e r e n c e in the r e d o x p r o p e r t i e s o f the p r o t e i n - h o u n d T T Q is attribu t e d to the accessibility o f only o n e of the T T Q carbonyls to solvent in MADH. In the r e d u c e d f o r m , the o t h e r quinol oxygen at C7 is shielded fl-om solvent a n d h y d r o g e n - b o n d e d to an a m i d e h y d r o g e n o f ttle p o l y p e p t i d e b a c k b o n e (see Fig. 11). Thus, the C7 oxygen is n o t protoh a t e d in the enzyme. E x a m i n a t i o n o f the e x t e n t to which the d i s p r o p o r -

1] 8

VICTORL. DAVIDSON

tionation of the MADH semiquinone occurred as a function of pH indicated that the equilibrium concentration of serniquinone increased with pH (Zhu and Davidson, 1998b). This indicates that the single proton transfer that is associated with the two-electron oxidation-reduction reaction is linked to the semiquinone/quinol couple. Therefore, the quinol is singly protonated and the semiquinone is unprotonated and anionic (Fig. 13). Kinetic studies have determined that the Em values at pH 7.5 for the one-electron couples are oxidized/semiquinone = +14 mV and semiquinone/reduced = +190 mV (Brooks and Davidson, 1994b). The substitution of N for O in the aminoquinol and iminosemiquinone appears to increase the F~ values by approximately 40 mV (Itoh et al., 1995; Bishop and Davidson, 1998). The unusual property of TTQ enzymes of exhibiting the anionic forms of the quinol and semiquinone is important for the reaction mechanism of MADH because it allows stabilization of physiologically important N-quinol and N-semiquinone reaction intermediates (see Sections V, C, 4 and 5). 3. Electron Paramagnetic Resonance (EPR) Studies of M A D H Semiquinones Electronic properties of the O-semiquinone (Warncke et al., 1995) and N-semiquinone (Warncke et al., 1993; Singh et al., 2000) redox forms of P. denitrificans MADH have been characterized by EPR, electron double nuclear resonance (ENDOR), and electron spin echo envelope modulation (ESEEM) spectroscopies. The EPR spectrum of the TTQ O-semiquinone form of MADH, which is prepared by dithionite reduction of the enzyme, was found to differ substantially from that observed for the N-semiquinone form, which is derived from substratereduced MADH. ESEEM data provided definitive evidence that the substrate-derived nitrogen is covalently bound to TTQ when the cofactor is in its one-electron reduced form, and that it has an imine-like structure. The intensities of the modulations also confirmed that the N-semiquinone generated in vitro by the light-induced oxidation method of Zhu and Davidson (1998a) results in a homogeneous preparation of the radical. A comparison of the N hyperfine and nuclear quadrupole couplings measured for the N-semiquinone, with those measured for the O-semiquinone, showed that a significant change occurs in the highest occupied molecular orbital when substrate nitrogen is bound (Singh et al., 2000). This may be related to the different redox and electron transfer properties of these two semiquinone forms. It is also interesting to note that the EPR and ESEEM studies revealed differences in the electronic structures of the O-serniquinone and N-semiquinone forms of MADH that were not apparent from comparison of their visible absorption spectra.

PQQ AND TTQ

[ I t}

--H

B2H

B 1 :..~ ~H 3

--~"-

NH+

0

~ (CH3'~"~ :B 3

CH3

t NH 2 B7:

M+

BB:

O-

- p--

NH

O-

I"~H B 6

)

/~/CH 2 HO

- ....

H20

~1

-

B4 J ~ C H

2

H20

BS: 2'"1 °x ) ~ / H 2 0 2AI41 r g d

HzO ~,~ N H ; B9 ~:

~ 0

HBIO

HO ~ ~NH2 0 Bll :

0

0

HBI2

FI(;. 14. Proposed chemical reaction mechanism for the conversion of methytamiutto formaldehyde plus ammonia by methylamine dehydrogenase. Only the quinone p(,tion of TTQ is shown. B] to BI2 represent active-site residues that may flmction as ge~eral acids or bases in the reaction mechanism. AMI represents amicyanin and M~ is a monovalent cation. The details of the reaction mechanisnrs are presented in the text.

C. Chemical Reaction Mechanism of M A D H A d e t a i l e d c h e m i c a l r e a c t i o n m e c h a n i s m f o r t h e overall o x i d a t i o u r e d u c t i o n r e a c t i o n o f M A D H with m e t h y l a m i n e a n d a m i c y a n i n , b a s e d o n r e s u l t s o f s t u d i e s o f t h e M A D H f r o m P. denitrificans, is s h o w n i n Fig. 14. T h e r e l e v a n t d e t a i l s o f e a c h r e a c t i o n s t e p are d i s c u s s e d below.

120

VICTOR L. DAVIDSON

1. Formation of the Enzyme-Substrate Complex The initial step in the oxidative deamination of methylamine by MADH is the formation of a covalent Schiff base imine adduct between the amino nitrogen of the substrate and the C6 carbon of TTQ. Since methylamine has a P/£a value of 10.6, it is likely that an active site residue is required to bind and deprotonate the substrate methylamm o n i u m to generate the neutral methylamine for nucleophilic attack of the C6 carbonyl carbon. This will initially form a carbinolamine intermediate that is dehydrated to yield the imine. A consequence of the susceptibility of the C6 carbonyl of T T Q to nucleophilic attack by amines is that all T T Q enzymes are irreversibly inactivated by a class of compounds with the general structure NH2-NHR (Kenny and McIntire, 1983; Davidson and Jones, 1992). This includes hydrazine, phenylhydrazine, semicarbazide, and aminoguanidine. Reaction with these compounds yielded relatively unreactive hydrazone adducts of TTQ. Hydroxylamine (NHz-OH) inhibits in a similar fashion, also yielding a covalent adduct of TTQ. Since the reactions of the hydrazines with the reactive carbonyl of T T Q proceed no farther than the initial imine formation, it has been possible using substituted phenylhydrazines to elucidate the factors that control the initial binding of the amine to T T Q in MADH (Davidson and Jones, 1995a). These inactivators have also been useful for identifying the reactive portion of the T T Q prosthetic group. Crystallographic analyses of MADH that had been treated with either methylhydrazine or 2,2,2-trifluoroethylhydrazine provided definitive evidence that the C6 carbonyl is the reactive site of T T Q in MADH (Huizinga et al., 1992). 2. Conversion to the Enzyme-Product Complex Conversion of the oxidized TTQ-substrate adduct to the reduced TTQ-product adduct is initiated by the abstraction of a proton from the methyl carbon of the substrate by an active-site basic residue. This occurs concomitant with the reduction of T T Q (Brooks et al., 1993). This reaction step has been studied in detail by characterizing deuterium kinetic isotope effects for the reaction with methylamine, and the kinetics of the reactions of MADH with alternative substrates. a. Kinetic Isotope Effects. When CD~NH2 was used as a substrate for MADH, a deuterium kinetic isotope effect of 17.2 was measured for the rate constant for reduction of T T Q (Brooks et al., 1993). Correction for possible contributions to this value from secondary isotope effects from the other two methyl deuteriums yielded a range of 9.3 to 17.2 for the true primary kinetic isotope effect. This indicates that the proton

PQQANDTTQ

121

abstraction step is the slowest of the reaction steps leading to T T Q reduction, and that the imine intermediate must accumulate prior to the hydrogen abstraction step. The deuterium kinetic isotope effect that was measured in steady-state kinetic experiments was 3.0 (Davidson, 1989), indicating that this proton abstraction is not the rate-determining step in the overall oxidation-reduction reaction. The reduction by dopamine of the T T Q prosthetic group of the other TTQ-dependent enzyme, aromatic amine dehydrogenase, also exhibited an unusually large deuterium kinetic isotope effect of 8.6 to 11.7 (Hyun and Davidson, 1995a). The magnitudes of these primary deuterium kinetic isotope effects for the two TTQ-dependent enzymes appear to exceed the semiclassical limit for a hydrogen abstraction reaction (Klinman, 1978). A similar large primary deuterium kinetic isotope effect in the range of 9.6 to 13.5 was measured for the hydrogen abstraction step in the reaction catalyzed by another quinoprotein, the topaquinone-containing bovine plasma amine oxidase (Palcic and Klimnan, 1983). For that enzyme, it was subsequently shown that the large isotope effec~ could be explained by a mechanism involving quantum mechanical proton tunneling (Grant and Klinman, 1989). The data obtained tier the T T Q enzymes suggest the possibility that similar quantum mechanical effects may also play a role in the hydrogen abstraction step of the reactions catalyzed by this class of enzvmes. b. Reactions with Alternative Substrates. It is generally believed that benzylamines are not substrates for MADH. Benzylamines do, however. stoichiometrically reduce T T Q and appear to act as competitive inhibitors ofmethylamine oxidation by MADH (Davidson el al., 1992b). Although the affinity of MADH for benzylamines is weak compared to methylamine, it was possible to study the reactions of a series of/)-substituted benzylamines with MADH. These data were used to construct H a m m e t t plots to ascertain structure-activity correlations (Hansch el al., 1991). Plots of the limiting first-order rate constant for T T Q reduction (k,-ed) and/~t against substituent constants, which reflected either res(~nance or field/inductive parameters for each p-substituent, indicated that the magnitude of h:+ was primarily influenced by field/inductive eftects whereas/~t was primarily influenced bv resonance effects. This is reasonable as resonance contributions would clearly stabilize the imine intermediate. The Ki values that were obtained from steady-state kinetic experiments, in which benzylamines were used as competitixe inhibitors of methylamine, correlated strongly with the Ka values that were obtained from rapid kinetic experiments. A positive slope of the H a m m e t t plot of k,-~a was obtained that is diagnostic of a carbanionic intermediate, and consistent with the mechanism of an active-siw

122

VICTOR L. DAVIDSON

nucleophile abstracting a proton from the methyl carbon, thus forming a carbanionic intermediate concomitant with the reduction of the TTQ prosthetic group. A similar pattern of results and conclusions were obtained from analogous studies of the other TTQ-dependent enzyme, aromatic amine dehydrogenase (Hyun and Davidson, 1995b). Results similar to those described above for the benzylamines were also obtained with allylamine (Davidson et al., 1995a). Allylamine also did not serve as substrate for MADH in a steady-state assay of activity, and appeared to act as a competitive inhibitor. However, transient kinetic studies revealed that allylamine stoichiometrically and rapidly reduced TTQ. The rate of T T Q reduction by allylamine was actually greater than the rate of reduction by methylamine. These data were explained by a kinetic mechanism in which allylamine and methylamine are alternative substrates for MADH. The apparent competitive inhibition by allylamine was due to a very slow rate of release of the aldehyde product (discussed in Section V, C, 3). 3. Release of the Aldehyde Product During the reduction of TTO~ the imine bond between the substrate nitrogen and C6 of TTQ is converted to a single bond, and a new imine bond forms between the substrate nitrogen and the methyl carbon. The latter bond is hydrolyzed to release the formaldehyde product and yield a reduced aminoquinol reaction intermediate (Bishop et al., 1996b). Activesite amino acid residues are presumably necessary to coordinate and activate water for nucleophilic attack of the imine carbon. Combined steady-state and transient kinetic studies of MADH with methylamine and alternative substrates indicate that the release of the aldehyde product is the rate-determining reaction step in the steady state. Steady-state and transient kinetic studies revealed that the apparent competitive inhibition by allylamine is due to a very slow rate of release of the acrolein, aldehyde product, 0.28 s-1 relative to 19 s-1 for release of the formaldehyde product of the oxidative deamination of methylamine. The latter Value is approximately that of kcat for the steady-state reaction of methylamine and phenazine ethosulfate with MADH (Davidson, 1989). The hydrolysis of the enzyme-product imine intermediate in the reaction with allylamine is evidently m u c h slower than the hydrolysis of this intermediate in the reaction with methylamine. This is likely a consequence of the extended conjugation that is provided by the allylic group relative to the methyl group. Similarly, the apparent competitive inhibition of MADH by benzylamines may be attributed to product release that is so slow that one is unable to detect significant turnover of the enzyme in the steady-state

VQ(2AXDTTQ

123

assay with phenazine ethosulfate. The r e d u c e d enzyme-product complex derived from benzylamines will be stabilized to a greater extent by the benzyl moiety attached to the methylene carbon relative to a simple hydrogen. Electrons may be delocalized in a m a n n e r that significantly lowers the relative concentration of the tautomeric form of the intermediate, which is susceptible to hydrolysis resulting in verx slow release of the aldehyde p r o d u c t relative to the reaction with aliphatic substrates.

4. Formation of a Stable Aminoquinol MADH The enzyme form that results from the release of the aldehyde product is a stable aminoquinol. The existence of a stable aminoquinol form of T T Q during the catalytic cycle of MADH was a matter of some controversy. Its identity was confirmed by NMR analysis of the reaction of MADH with methylamine (Bishop et al., 1996b). 13C- and 15N-NMR studies of the reactions of MADH with 13C- and ~SN-labeled methylamine demonstrated that the products of the reductire half-reaction are an equivalent of formaldehyde hydrate and a reduced aminoquinol form of T T Q that possesses covalently bound snbstrate-derived nitrogen. When the reaction was monitored by NMR. the reduction of MADH by 15N-enriched methylammonium chloride resulted in the formation of a new nitrogen signal that exhibited a chemical shift characteristic of a nitrogen that is covalently bound to an aromatic ring (i.e., an aminoquinol). This signal appeared optimally with short pulse delay, which is consistent with the nitrogen being attached to a rigid protein matrix. This signal was retained after exhaustive dialysis and only disappeared after the subsequent reoxidation of the reduced enzyme. The oxidation of this aminoquinol resulted in the appearance of a signal with a chemical shift and relaxation time chara(teristic of fi'ee a n n n o n i u m ion. Ve O, similar results were obtained in studies of the reaction of aromatic amine dehydrogenase with ~!~(;-and 15N-labeled amine substrates (Bishop et al., 1998).

5. Reoxidation of the Reduced Aminoquinol MADH to N-Semiquinone by A mi~ani~ MADH is reoxidized in two one-electron transfers to amicyanin molecules. The first electron transfer step requires the presence of a monovalent cation that is proposed to be coordinated by an active-site residue (Bishop and Davidson, 1997). Another general base is required to deprotonate the amino nitrogen and thus activate this intermediate for electron transfer to amicyanin (Bishop and Davidson, 1997). The details of the mechanism of this reaction step are discussed later in Section V, I), 2, b.

124

vlcxol~ L. DAVIDSON

The substrate-derived amino nitrogen remains bound to T-FQ after the first electron transfer to yield an iminosemiquinone form of TTQ.

6. Oxidation of N-Semiquinone MADH by Amicyanin Oxidation of the iminosemiquinone intermediate yields an oxidized imine form of T-FQ with substrate-derived nitrogen still bound to the C6 carbon. In the absence of another molecule of substrate, this imine intermediate will be hydrolyzed to the quinone in a relatively slow reaction (see Fig. 14). Alternatively, in the steady state, the amino nitrogen of another molecule of substrate, rather than water, may react directly with the iminoquinone to form the next enzyme-substrate adduct with concomitant release of the ammonia product (Fig. 15) (Zhu and Davidson, 1999). The visible absorption spectrum of the iminoquinone intermediate observed in these transient kinetic studies is similar to that reported for some ammonia adducts of MADH. A m m o n i u m has been shown to be a reversible competitive inhibitor of MADHs and exhibit a Ki of approximately 20 mM (McIntire, 1987; Davidson and Jones, 1992). For the MADH from bacterium W3A1, a m m o n i u m also functions as an activator at lower concentrations with a KA of 2 mM (McIntire, 1987). Addition of a m m o n i u m salts to oxidized MADH has also been shown to cause perturbations of its absorption spectrum (Kuusk and McIntire, 1994; Goren and Duine, 1994; Davidson et al., 1995b). A titration of MADH from bacterium W3A1 with NH4CI + NH3 was biphasic, and it was concluded that the first phase reflected binding of ammonia to T T Q to p r o d u c e an iminoquinone (Kuusk and McIntire, 1994). The absorption spectrum of that species was qualitatively similar to the spectrum of the putative iminoquinone intermediate that was observed in transient kinetic studies with the P. denitrificans enzyme (VI in Fig. 15) (Zhu and Davidson, 1999). A qualitatively similar result was observed with MADH from T. versutus (Goren and Duine, 1994). The other T T Q enzyme, aromatic amine dehydrogenase, showed a similar spectral perturbation on addition of ammonia that was also concluded to reflect binding of ammonia to T T Q to form an iminoquinone (Zhu and Davidson, 1998c). It is difficult to compare these results because the enzymes from the different sources have slightly different absorption maxima and the ammonia titrations were p e r f o r m e d at different p H values, which can influence the spectral properties of MADH in the presence of monovalent cations (Kuusk and McIntire, 1994). However, these results strongly support the conclusion that the oxidized iminoquinone is the immediate product of the complete oxidation of the aminoquinol, and that the second product, ammonia, is released only after complete reoxidation of TTQ.

PQQAND TTQ

125

I NH3

~, ~

H20..,-~'""'" Vl

/

O

CH3NH 2

O "'"'"~.

.-"

)-4"O HN

i

".

H20 ..

CH3NH2

'~

)

lI

Ha ~N

H--/C\H H :B

mired

1l

Amio×

Amiox

V

H

Amir~ + H*

O

I

IV

/ HCHO

HI

H20

N

O-

CH2 HB

F[(;. 15. Proposed chemical reaction mechanism for the stead}~state reaction of methylamine dehydrogenase with methylamine and amicyanin. Only the reactive portion of TTQ is shown. B represents an active-site residue, Kinetically distinguishable intermediate enzyme forms are labeled with Roman numerals: (I) the resting form of the enzyme, (II) the covalent enzyme-substrate complex, (IIl) the covalent enzymeproduct complex, (IV) the N-quinol, (V) the N-semiquinone, (VI) tile N-quinone.

7. Possible Roles of Active-Site Residues in the Catalytic Mechanism The detailed reaction mechanism shown in Fig. 14 lists as many as 12 roles for active-site residues in the overall reaction mechanism. These are indicated as B1-B12. These residues function primarily as general acids and bases, but are also required for monovalent cation binding and possibly proper orientation of substrate, water, and reaction intermediates. It is likely that multiple roles may be performed by a single residue so that less than 12 active-site residues would be sufficient to catalyze the complete oxidation-reduction reaction. The crystal structure of MADH reveals the presence of four amino acid residues of the ~ subunit in the active site that

126

VICTOR L. DAVIDSON

:P ~32

Asp 76

r 119

FIG. 16. Potentially reactive a m i n o acid residues in the active site of methylamine dehydrogenase. T h e structure is that of the enzyme from P. denitrificans (Chen et al., 1998; Protein Data Bank entry 2BBK).

could potentially participate in these reactions (Fig. 16). Another residue, Phe-55, of the ~ subunit is also located at the opening of the active site. Site-directed mutagenesis studies have shown that this residue plays a role in dictating the substrate specificity of MADH (Zhu et al., 2000a). D. TTQ in Electron Transfer MADH, amicyanin, and cytochrome c-551i from P. denitrificans form one of the best characterized electron transfer complexes of proteins. It has provided a powerful system with which to study mechanisms of interprotein electron transfer. Crystal structures have been determined for a binary protein complex of MADH and amicyanin (Chen et al., 1992), and a ternary protein complex of MADH, amicyanin, and cytochrome c-551i (Chen et al., 1994). The orientation of the three redox centers in the crystal structure are shown in Fig. 17: TTQ of MADH, copper of arnicyanin, and heine of cytochrome c-551i. In the crystalline state, the complex catalyzes methylamine oxidation and subsequent electron transfer from T T Q to heine via copper, as demon-

| 27

PQQ AND TTQ

~eme FI(;, 17. Orientation of redox cofactors in the methylarnine dehydrogenase-amicyanin-cytochrome c-551i complex. One half of the ctwstal structure of the ternaxT protein complex is shown. The direct distances between the cothctors (dashed lines) are 9,4 ]~ from TTQ to copper and 23 ,~ from copper to berne. The structure is that of the complex of proteins from R denitrificans (Chen et al., 1994; Protein Data Bank entry 2MTA).

strated by substrate-dependent spectral changes viewed by single crystal polarized absorption microspectroscopy (Merli et al., 1996). Results of site-directed mutagenesis studies confirmed that the site of interaction of MADH with amicyanin that is seen in the crystallized complex is the same as that used by the proteins when they interact in solution (Davidson et al., 1997; Zhu et al., 2000b). This is a physiologically relevant complex in which amicyanin is an obligatory mediator of electron transfer from MADH to the cytochrome. The amicyanin gene is located immediately downstream of that for MADH and inactivation of the former results in loss of the ability to grow on methylamine (van Spanning et al., 1990). MADH, amicyanin, and cytochrome c-551i are isolated as individual soluble proteins, but they must form a ternary complex to catalyze methylamine-dependent cytochrome c-551i reduction (Husain and Davidson, 1986; Gray et al., 1986, 1988; Davidson and Jones, 1991, 1995b). Although it is thermodynamically faw)rable, MADH does not

128

VICTOR L. DAVIDSON

reduce cytochrome c-551i in the absence of amicyanin because the proteins do not interact in a productive manner. Amicyanin will not donate electrons to cytochrome c-551i in the absence of MADH at physiologic p H because the redox potential of arnicyanin is more positive than that of the cytochrome. The redox properties of arnicyanin are altered on complex formation with MADH so as to facilitate the reaction (Zhu et al., 1998). Other structurally similar type I copper proteins, plastocyanin and azurin, do not effectively substitute for amicyanin (Gray et al., 1986; Hyun and Davidson, 1995c). A structural feature that distinguishes T T Q in MADH from that of many other redox cofactors is that it is able to physically link the active site of the enzyme to the protein surface. The phenyl portion of the indole side chain of Trp-108 breeches the surface of MADH and is only 9.4 ~, from the copper site of amicyanin when the MADH-amicyanin complex is formed (see Fig. 17). Thus, T T Q serves as a bridge between the chemical reactions that occur in the enzyme active site, and the electron transfer reactions that occur via the enzyme surface. This has allowed MADH to be used as a model for studying the interplay between catalysis and long-range electron transfer by a redox cofactor. Before discussing the mechanism of electron transfer from T T Q it is necessary to briefly review electron transfer theory.

1. Electron Transfer Theory The theoretical basis for what physical parameters control the rates of nonadiabatic electron transfer reactions is well established. Unlike adiabatic chemical reactions that involve the making and breaking of bonds, and proceed via a well-defined reaction coordinate, the substrates and products of a nonadiabatic protein electron transfer reaction are often chemically indistinguishable. For an adiabatic chemical reaction, the probability of the reaction occurring when the activation energy is achieved is approximately unity, but for a nonadiabatic reaction, the probability of the reaction occurring when the activation energy is achieved is much less than one. Nonadiabatic electron transfer theory (Marcus and Sutin, 1985) predicts that the rate of an electron transfer reaction (kET) will vary predictably with temperature (T), AG° (which is related to the redox potential difference between reactants), and donor-acceptor distance (r) according to Eqs. (4) and (5). 4n2HAB2 kET = h 4 ~ T exp [-(AG ° + )~)2/4XRT]

(4)

kET = k0 exp [-J3(r-r0) ] exp [-(AGO + ~,)2/4~RT]

(5)

PQQ AND TTQ

129

HAB is the electronic coupling matrix element. It is the degree of nonadiabaticity (i.e., the probability of the reaction occurring in the transition state) and is related to the electronic coupling between reactants and products in the transition state. The reorganizational energy ()v) is the energy n e e d e d to deform the nuclear configuration from the reactant to the p r o d u c t state, and is composed of two components. The inner sphere reorganizational energy ()vi) reflects r e d o x - d e p e n d e n t nuclear perturbations of the redox centers, such as changes in b o n d lengths. The outer sphere reorganizational energy ()Vo) reflects changes in the surrounding medium, such as changes in solvent orientation and protein polarization. The factor ~ is related to the nature of the intervening m e d i u m between redox centers. Detailed discussions of the mathematical and physical meaning of HAB and )v may be f o u n d in a n u m b e r of reviews of electron transfer theory (Marcus and Sutin, 1985; McLendon, 1988; Moser et al., 1992; Gray and Winkler, 1996; Davidson, 2000). The other terms in Eqs. (4) and (5) are Planck's constant (h), the gas constant (R), the characteristic frequency of the nuclei (ko), and the close contact distance (~;,), which is usually assigned a value of 3.0 A. With MADH and amicyanin it has been possible to test the applicability of Eqs. (4) and (5) to the study of interprotein electron-transfer reactions. A significant problem in applying electron-transler theory to longrange electron-transfer reactions that occur in and between proteins is that it is often difficult to ascertain whether or not the measured rate of the redox reaction is a true /~T. For a simple bimolecular protein electron-transfer reaction, the following three-step reaction scheme may be envisioned (Eq. 6). Kd Aox + Bred , " Aox/Bred

-,

k x [A,,x/Bred]* bET

In this scheme, some non-electron transfer reaction step with a forward rate constant of kx and an equilibrium constant K× (kx/It-x), occurs after binding of the two redox proteins and is required to activate the protein complex for electron transfer. If kx < kFT, then the reaction is defined as being gated (Hoffman and Ratner, 1987; Davidson, 1996). If kx > ~T, but I% << 1 for the prerequisite adiabatic reaction step, then the electron-transfer reaction is defined as kinetically coupled (Harris et al., 1994; Davidson, 1996). In this case, the rate constant for the conversion of Aox/Bred to Ared/Box will not be kET, but hE-r'K×. With the MADH system it has been possible to develop methods tor distinguish-

130

VICTOR L. DAVIDSON

ing between nonadiabatic and gated electron-transfer reactions and also to characterize a detailed chemical reaction mechanism for a gated electron-transfer reaction involving the redox reaction of one of the redox forms of TTQ.

2. Electron Transferfrom Different Redox Forms of MADH to Amicyanin It is possible to study electron transfer from several different redox forms of MADH to amicyanin (see Fig. 13). Because T T Q is a twoelectron carrier and the type I copper is a one-electron carrier, two sequential oxidations of fully r e d u c e d T T Q by amicyanins are required to completely reoxidize MADH. O-quinol and O-semiq u i n o n e forms of MADH may be g e n e r a t e d by reduction by dithionite (Husain et al., 1987). As discussed earlier (Section V, C, 4), the p r o d u c t of the reduction of MADH by the substrate amine is an Nquinol that retains the covalently b o u n d substrate-derived amino group after release of the aldehyde product. An N-semiquinone is the p r o d u c t of the first one-electron oxidation of the N-quinol. Each of these forms may also be g e n e r a t e d in vitro (Bishop et al., 1996b; Zhu and Davidson, 1998a). Electron transfer between MADH and amicyanin was studied in solution by stopped-flow spectroscopy. For each form it was possible to examine the d e p e n d e n c e of the rate of the redox reaction on t e m p e r a t u r e and analyze the data according to Eqs. (4) and (5) (Table III). It was also possible to examine the d e p e n d e n c e of the rate of the redox reaction on AG° because the zMY~n is different for the reaction of each different redox form of MADH with amicyanin.

a. Nonadiabatic Electron Transferfrom TTQ to Copp~ The reactions of the O-quinol, O-semiquinone, and N-semiquinone forms of MADH with amicyanin exhibited a predictable d e p e n d e n c e on AG° (Brooks and Davidson, 1994b; Bishop and Davidson, 1998). Analysis of the AGOd e p e n d e n c e of kET yielded values of ~, and HAB that were identical to those obtained from analysis of the temperature dependencies of kET for the reactions with amicyanin of the O-quinol and N-semiquinone (Brooks and Davidson, 1994a, 1994b; Bishop and Davidson, 1998). These analyses also predicted an ET distance that closely matched that seen in the crystal structure (see Table III). b. Gated Electron Transferfrom TTQ to Copp~ In contrast to the nonadiabatic electron transfer reactions from MADH to amicyanin, analysis of the temperature d e p e n d e n c e of the reaction between amicyanin and the N-quinol form of MADH yielded unreasonable values of ~ and HAB

131

PQQ AND TTQ

TABLE I l i

Parametersfor the Reduction of Amicyanb~ by Different Redox l'brms of MAI)H O-quinol

O-semiquinone

N-quinol

N-semiquinom'

kl.Tr in 10 mM KPi + 0.2 M KCI, pH 7.5 (s-I)

12

>500

130

51

Kinetic solvent isotope effect (He°k/t)e°k) at pH 7.5

1.5

ND"

6.5

1,8

2.3_+0.1

2.3_+ O,l

3.4_+0.1

2.4_+0,1

12_+ 7

12 +_ 7

>20,000

13 +- t

9.6 + 0.7

9.6 +_0.7

<0

9.4 _~. 1.2

Electron transi~er Brooks and Davidson (1994a,b)

Electron tl-anslel Brooks and [)avidson (1994b)

~. (cV) t['~B ( c m q )

r (A, f~,- ~-1) Rate-limiting step Relerencc

Proton Ii3.11sfeiBislmp and Davidson ( I ,q95)

Electron IFansl¢.l Bishop aim Daxi(lson (1998)

" ND, not determined.

and a negative value for electron-transfer distance (Bishop and Davidson, 1995) (see Table III). The rate of this reaction also exhibited a large deuterium kinetic solvent isotope effect and p r o n o u n c e d dependence on pH, indicating that the adiabatic reaction step that gates the electron-transfer reaction from N-quinol MADH is the transfer of an exchangeable proton. Furthermore, the rate of the gated electrontransfer reaction from the N-quinol MADH to amicyanin was dependent on pH and the concentration of monovalent cations (Bishop and Davidson, 1997). The proposed mechanism fbr the cation and pH-dependent deprotonation of N-quinol T T Q that gates electron transfer from MADH to amicyanin is summarized in Fig. 18. In this model, binding of a monovalem cation (M +) plays an essential role in deprotonating N-quinol T T Q and activating it for rapid electron transfer to amicyanin. For M + to bind at the active site requires that an active-site base (X1 in Fig. 18, Bv in Fig. 14) provides a ligand in close proximi W the N-quinol nitrogen. The pH dependence of the reaction reflects the need to deprotonate this activesite residue so that it may bind M +. After binding M ~, TTQ-bound -NH,, is deprotonated by another active-site residue (X~ in Fig. 18, Bs in Fig. 14). It must be the amino group of the N-quinol that is deprotonated, rather than the hydroxyl group, since the C7 o~'gen is not protonated in the reduced state (Zhu and Davidson, 1998b). M + may interact with lhe

132

XIH

VICTOR L. DAVIDSON

H

L

.. Xl:,,M~

O'
~x~

H,~,,

H+

X2:

Xl:,,M+,, _

kET ... X~:',M~ X2H

X2H

FIG. 18. Proposed mechanism for monovalent cation-dependent electron transfer from aminoquinol methylamine dehydrogenase to amicyanin. Dashed lines indicate electrostatic and H-bonding interactions. X1 and X2 are ionizable active-site residues. This electron-transfer reaction is gated because the rate of the preceding protontransfer reaction (kpT) is slower than the rate of the electron-transfer reaction (kET). Details of this mechanism are discussed in the text, and in greater detail in Bishop and Davidson (1997).

-NH2 group via the lone pair of electrons on N and weaken the N - H bond to activate it for proton abstraction, stabilize the formation of a transition-state intermediate with partial negative charge, and stabilize the anionic product following deprotonation. After this rate-limiting deprotonation, rapid electron transfer occurs. Electron transfer from this activated intermediate is expected to be highly favored relative to electron transfer from the neutral aminoquinol because of the relatively electron-rich character of this activated intermediate. In the absence of M +, deprotonation of this group is much less favorable. This explains the large rate enhancement by M +. The immediate product of the electrontransfer reaction, an anionic radical, then rearranges to an imp nosemiquinone. This mechanism involving the deprotonation of T r Q - b o u n d -NH2 is also consistent with results of ESEEM experiments that indicated that the substrate-derived nitrogen on the N-semiquinone exists as an imine with the lone pair of electrons that reside in its sp 2 hybridized orbital involved in the formation of a hydrogen bond with a nearby proton donor (Singh et al., 2000). This gating p h e n o m e n o n is only observed with the aminoquinol form of MADH. This demonstrates that the covalent incorporation of substrate-derived N into reduced T T Q during the reductive half-reaction has a profound effect on the rates and regulation of the electron transfer reaction from MADH to amicyanin (see Table III). Thus, in the MADH-amicyanin complex it is possible to modulate the rate for the electron-transfer reaction, which occurs over the same pathway or distance between T T Q and copper, either by changing z~G~ for the reaction or, in the case of the N-quinol, by modifying the T T Q cofactor so that a chemical reaction (i.e., deprotonation) becomes rate limiting for electron transfer.

PQQAND TTQ

133

A similar pattern of nonadiabatic electron transfer from O-quinol T T Q and gated electron transfer from N-quinol T T Q is seen in the reaction of aromatic amine dehydrogenase with its electron acceptor, the type I copper protein azurin (Hyun et al., 1999). In this system the electron-transfer reaction from the aminoquinonol is also gated by the transfer of a solvent-exchangeable proton.

E. Roles of p H and Cations In addition to the effects of monovalent cations on regulating the process of gated electron transfer from N-quinol T T Q to amicyanin, monovalent cations may play additional roles in the reactions of MADH, Further evidence for the interaction of monovalent cations with T T Q at the active site of MADH comes from studies of the effects of pH and monovalent cations on the spectral properties of oxidized MADH. Two types of cation binding sites have been described by Kuusk and McIntire (1994). One is more specific for larger cations such as Cs+, Rb +, and NH4 +. The other is more specific for smaller cations such as K+ and Na +. Consistent with the results of these studies on the perturbations of the absorption spectrum of MADH by these monovalent cations, monovalent cation-bound forms of MADH have been characterized by X-ray crystallography (Labesse et al., 1998). MADH crystals were incubated with Cs+, and two Cs+ ions were identified in the active site. Microspectrophotometry of the crystals indicated that the binding of the cations in the crystal caused the same spectral perturbations that were observed in solution (Labesse et al., 1998). Monovalent cation-dependent spectral perturbations have also been observed for the TTQ-dependent aromatic amine dehydrogenase (Zhu and Davidson, 1998c). TTQ in that enzyme may be converted to a hydroxide adduct by incubation at alkaline pH in the presence of monovalent metallic cations, such as Na + and K+. Although there is no evidence that this is a catalytically relevant form, it can be distinguished spectroscopically. These observations were important in establishing that the actiw, sites of these TTQ enzymes bind monovalent cations. These data are also consistent with the proposed physiologic role for a monovalent cation at the active site of facilitating the physiologic electron-transfer reaction fi'om N-quinone TTQ to copper (see section V, D, 2, b).

E Biosynthesis of TTQ and MADH T T Q is formed by a posttranslational modification of two tryptophan residues (Mclntire et al., 1991). In P. de~dtr!ficans, these residues are

134

VICTOR L. DAVIDSON

TABLE IV The m a u Gene Cluster of P. denitrificans

Features inferred from s e q u e n c e

Gene

Function

MauR

Regulation

MauF

Unknown

MauB

MADH large subunit

Structure

MauE

Unknown

MauD

Required for MADH biosynthesis

Reference

LysR-type transcription activator

No

van S p a n n i n g et al. (1994)

--

Yes

van d e r Palen et al. (1995)

Yes

Chistoserdov et al. (1992)

M e m b r a n e protein

Yes

van d e r Palen et al. (1997)

Unknown

Cys-X-X-Cys m o t i f for disulfide b o n d formation/ isomerization

Yes

van der Palen et al. (1997)

MauA

MADH small subunit

Structure

Yes

Chistoserdov et al. (1992)

MauC

Anticyanin

Structure

No

van S p a n n i n g et al. (1990)

mauJ

Unknown

--

No

van d e r Palen et al. (1995)

mauG

Unknown

Similarity to peroxidases

Yes

van d e r Palen et al. (1995)

mauM MauN

Unknown Unknown

Similarity to ferredoxins Similarity to ferredoxins

No No

van d e r Palen et al. (1995) van d e r Palen et al. (1995)

Trp-57 and Trp-108. Two atoms of oxygen are incorporated into the indole ring of Trp-57 and a covalent b o n d is formed between the indole rings of the two tryptophan residues (see Fig. 1). The mechanism by which this occurs has not yet been elucidated, but it requires the action of other proteins that are subject to the same genetic regulation as the structural genes for the enzyme. The methylamine utilization (mau) gene cluster contains several genes, at least four of which encode proteins that are required for biosynthesis of MADH, in addition to the two MADH structural genes (van der Palen et al., 1995; Graichen et al., 1999). The mau gene cluster from P. denitrificans is described in Table IV. Similar mau gene clusters have been characterized from Methylobacterium extorquens AM1 (Chistoserdov et al., 1994a) and Methylophilus methylotrophus W3A1 (Chistoserdov et al., 1994b), although the latter lacks the mauC gene that encodes amicyanin. None of the accessory gene products that are absolutely required for MADH synthesis have been isolated, but from the d e d u c e d sequences of these four genes; the mauE product appears to be a m e m b r a n e protein, the mauD product may be involved in disulfide b o n d processing, the mauG product may

PQQANDTTQ

135

be a diheme-bearing peroxidase, and m a u F s h o w s no sequence homology to any class of proteins. These gene products must play essential roles in translocation of MADH subunits across the cell m e m b r a n e to the periplasm, formation of the six disulfide bonds that are present in the TTQ-bearing ]3-subunit, and oxygenation reactions that are required for T T Q biosynthesis. Furthermore, the m a u B gene that encodes the MADH ~ subunit possesses an unusual 57-residue long signal sequence (Chistoserdov et al., 1992, 1994a), which exhibits a double arginine motif that is typically translocated by the gene products of the special tatABCD operon (Sargent et al., 1998). It has been proposed thai signal sequences such as this may be characteristic of periplasmic proteins that possess complex redox cofactors (Berks, 1996). It is interesting to compare the biosynthesis of MADH with that of other quinoproteins. M t h o u g h T T Q is structurally sinfilar to PQO_~ the cofactors are clearly derived from completely different processes. T T Q is similar to the topaquinone prosthetic group (lanes et al., 1990) of the amine oxidases in that it is a quinone derived by posttranslational modification of an amino acid on the polypeptide chain. However, the modification of tyrosine to yield topaquinone is an autocatalytic process requiring only the presence of copper and oxygen (Matsuzaki et al., 1994; Cai and Klinman, 1994). MADH contains no redox-active metal and the modification of two tryptophans to yield T T Q appears Io require several accessory proteins.

VI. SUMMARYAND PERSPECTI\,~S

The chemical reaction mechanisms for catalysis by PQQ- and TTQd e p e n d e n t enzymes are very similar to those used by other enzymes that possess carbonyl cofactors such as pyridoxal phosphate-, topaquinone-, and pyruvoyl-dependent enzymes. Pyridoxal phosphate is widely distributed in nature. While topaquinone is less common, it may be synthesized autocatalytically in the presence of copper. As discussed in this review, the biosyntheses of P Q Q and T T Q are relatively complex processes. Each requires participation of several genes and gene products. This suggests that in the organisms that use these cofactors, P Q Q and T T Q must play very specific and critical roles. Otherwise it is difficult to imagine how and why these complex biosynthetic machineries evolved and were retained. In speculating as to what the special need for P Q Q and T T Q might be, it is noteworthy that these cofactors participate not only in catalysis, but also in long-range electron transfer. The other cofactors that could

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perform the catalytic functions of PQQ and TTQ do not participate in long-range electron transfer. Pyridoxal phosphate does not normally participate in redox reactions. Topaquinone-dependent enzymes are oxidases, and while redox active, they transfer electrons directly to the electron acceptor, oxygen, at the enzyme active site. Another curious feature of these quinoprotein dehydrogenases is that the host organisms that utilize PQQ-dependent enzymes use T T Q as the prosthetic group for the amine dehydrogenases. Most, if not all, bacteria that use TTQ-dependent amine dehydrogenase also synthesize PQQ-dependent enzymes. The results of model studies have shown that free PQQ can catalyze the oxidation of primary amines (Rodriguez and Bruice, 1989; Itoh et al., 1991). However, PQQ is used in these bacteria only as the cofactor for alcohol and sugar dehydrogenase, while T T Q is exclusively used as the prosthetic group for the amine dehydrogenases. Although the study of PQQ- and TTQ-dependent enzymes has raised some intriguing philosophical questions, more importantly it has also provided scientists with valuable tools for addressing several fundamental issues. As discussed in this review, the studies of these two classes of enzymes are allowing us to learn a great deal about mechanisms of carbonyl catalysis by enzymes, mechanisms of longrange interprotein electron transfer, and mechanisms of protein biosynthesis and posttranslational modification. With the recent determinations of high-resolution crystal structures of more enzymes, and the development of systems for expression and sitedirected mutagenesis of these enzymes, further insight into these fundamental biochemical processes is anticipated.

ACKNOWLEDGMENTS Work performed in this laboratory has been supported by NIH grant GM-41574. I am also very grateful for the contributions of several former and current members of this laboratory and collaborators whose names are included in our joint publications that are cited here.

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